2Adriaan O. Kraaijeveld1,2, Saskia C.A. de Jager2, Theo J.C. van Berkel2,

Erik A.L. Biessen2 and J.Wouter Jukema1

Chemokines and Atherosclerotic Plaque Progression:

Towards Therapeutic Targeting?

Abstract: Atherosclerosis is currently viewed as an in�lammatory disease in which the initiation and progression of the atherosclerotic plaque towards a rupture prone, unstable plaque is driven by leukocyte recruitment mediated by various in�lammatory mediators. Recently, interest in chemotactic cytokines or chemokines with regard to atherosclersis has been growing as chemokines mediate the in�lux of leukocytes that is typical of atherothrombosis. The activity of the majority of chemokines is overlapping and che-mokines are not only produced by the various cellular constituents of the atherosclero- tic plaque but also by activated platelets. Consequently, the direct in�luence of individu-al chemokines on plaque destabilisation and rupture is widespread and rather unclear. Experimental research has already established the role of a number of chemokines in advanced atherosclerosis. Nevertheless, given the complexity and size of the chemo-kine family, further screening of cardiovascular disease for chemokine level and genetic polymorphisms for chemokines will be warranted as the search for viable biomarkers of plaque destabilization as well as novel therapeutic targets for speci�ic atheroregres-sive therapeutic compounds is ongoing. With regard to the latter, clinical trials with spe-ci�ic chemokine inhibitory strategies, like chemokine receptor antagonists, are already underway in other in�lammatory disorders. Summarizing, chemokine inhibition likely constitutes an important therapeutic option next to already established drugs in the management of cardiovascular disease.

Curr Pharm Des. 2007;13(10):1039-52

29

Introduction

Cardiovascular disease (CVD) is the largest contributor to morbidity and mortality in the industrialized world. In 2002, the prevalence of CVD in the American population was 34.2%, accounting for estimated direct and indirect health care costs of 393.5 bil-lion dollars in 2005 and claiming 2600 deaths each day1. The largest contributor to CVD, among others, is the slumbering process of atherosclerosis and the culprit for its clinical consequences, atherothrombosis.

Over the past 20 years, our knowledge on the pathophysiology of athero-sclerosis and, to a lesser extent atherothrombosis, has increased tremendously. Vari-ous in-depth reviews summarize a very compelling body of evidence indicating that atherosclerosis is a chronic in�lammatory process2, 3, as �irst postulated by Ross4. While current paradigm views atherosclerosis as an in�lammatory, progressive disease of com-plex aetiology, hyperlipidaemia, among other risk factors, is intimately linked to vascu-lar wall in�lammation and intimal stenosis. Interestingly, clinical events were found to be associated with the composition and vulnerability of the plaque rather than with the degree of stenosis5. Together with the observation on atherosclerotic plaques obtained at autopsy that thin capped �ibroatheroma are mechanically weaker than those with thicker caps, these �inding have prompted the introduction of the vulnerable plaque concept6-8. In brief, atherosclerotic lesions are formed in the intima of the vessel wall and what begins with an asymptomatic intimal fatty streak gradually becomes a more advanced lesion through the recruitment and in�lux of various in�lammatory cell types, migration of smooth muscle cells (SMCs) and the formation of a lipid-rich necrotic core with an overlying �ibrous cap. Eventually, the plaque will advance to an unstable vulner-able plaque phenotype or thin cap �ibroatheroma9, 10. The progression of an advanced to a vulnerable plaque is suggested to result from a local dysbalance in cellular as well as extracellular composition of the plaque leading to progressive erosion of the �ibrous cap and concomitant expansion and disintegration of the necrotic core. Ultimately, upon rupture of the latter plaque its highly thrombogenic content will be extruded to cause thrombus formation, arterial occlusion and ischemic symptoms or infarction. Thus, a thorough understanding of the molecular pathways underlying leukocyte in�lux in advanced and vulnerable plaques is from a clinical point of view of utmost importance.

The majority of studies have centred on addressing leukocyte in�lux in plaque initiation, establishing a prime role for selectin family members (i.e. E-selectin, L-selec-tin and P-selectin) in the capture, tethering and rolling of circulating monocytes onto the in�lamed endothelium and for endothelial adhesion molecules (e.g. ICAM-1, VCAM-1), which mediate leukocyte arrest by interacting with integrins on activated monocytes11,

12. Moreover these studies have revealed a key role of a speci�ic subclass of small chemo-tactic cytokines with strong leukocyte homing capacity, the chemokines, in this process. Chemokines were seen to contribute to the recruitment of leukocytes in initial stages of atherosclerosis by triggering chemotaxis and by assisting in arrest and diapedesis13-15. Given the clinical relevance of plaque rupture, leukocyte in�lux in advanced plaques has been subject of surprisingly few studies. Nevertheless it is conceivable that leukocyte in�lux in advanced plaques will differ in many aspects from that to in�lamed endothe-lium or initial plaques. The altered composition of advanced plaques likely translates in a changed panel of secretory mediators and chemokine receptors16. Moreover, the re-ported presence of vasa vasorum in close proximity to and neovessels within advanced lesions will certainly impact the pattern of leukocyte recruitment17, as these vessels will serve as an alternative entry site for circulating leukocytes.

While adequate (mouse) models for plaque initiation and progression are avail-able, there still is considerable debate on the relevance of models for studying (throm-botic) plaque rupture. Although plaque rupture have incidentally been observed in aged atherosclerotic mice, at speci�ic locations (i.e. the brachiocephalic artery) and after me-

Chapter 2

30

chanical or genetic manipulation18, 19, compelling evidence of thrombus formation at the site of presumed rupture is lacking, possibly due to the high �ibrinolytic propensity of mice20. Thus, until now a golden standard for (thrombotic) plaque rupture is still not available and by necessity we will have to live with surrogate rather than end-point markers of plaque rupture such as collagen content, cap thickness, lipid core content, buried caps, and intraplaque hemorrhage. Hence any data on effects of chemokines on plaque stability should be handled with considerable caution and can not be directly ex-trapolated to human situation. In this review, we will present our current knowledge on the role of chemokines in atherothrombosis and plaque stability with special emphasis on emerging therapeutic opportunities for stabilizing the vulnerable plaque.

ChemokinesChemokines are organised into 2 major (CC and CXC) and 2 minor (C and CX3C) families based on the N-terminal cysteine residues21. So far, approximately 50 chemokines have been described, as well as more than 20 of their seven transmembrane G-protein coupled receptors (GPCRs), while it has been shown that CC and CXC chemokine receptors can have multiple high af�inity ligands. Conversely, chemokines can bind various receptors as well, which makes chemokine activity very redundant and overlapping. Chemokines are key actors in leukocyte chemotaxis, leukocyte degranulation and mediator release, integrin activation during leukocyte-endothelial interaction and the induction of angio-genesis. Therefore, chemokines have a profound in�luence on various components of immunology and, as atherosclerosis is regarded as a chronic in�lammatory process, an increasing body of research is performed that underscore the in�luence of chemokines in atherothrombosis. From basic animal research as well as clinical research, 20 of these chemokines were reported to affect atherosclerosis (Tables 1, 2 and 3)22. Endothelial cells, vSMCs and macrophages express chemokine receptors, which make chemokines important players in plaque formation and destabilization by orchestrating selectin and integrin mediated monocyte recruitment, as well as supporting intimal migration of SMCs. As the relative contribution of these cell types changes during lesion progres-sion, the contribution of involved chemokines will likely change as well. Illustratively, Lutgens et al., using gene pro�iling techniques in ApoE-/- mice, showed a time dependent up-regulation of monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic pro-tein-5 (MCP-5), macrophage in�lammatory protein-1α (MIP-1α), MIP-1α and fractalkine with plaque progression, mainly in plaque macrophages. This upregulation was accom-panied by an increase in MCP-1 and MCP-5 serum levels.

A recent advance in the understanding of chemokine function and athero-thrombosis is their synergy with platelets. While platelets not only respond to and are activated by membrane-bound chemokines, they also release chemokines, such as PF4 (CXCL4) and RANTES (CCL5), and mediate deposition of these and other chemokines on the endothelium as recently reviewed14. Moreover, PF-4 even has an in�luence on lipoprotein metabolism modulating lipoprotein uptake by macrophages, illustrative of the pleiotropic effects of chemokines in atherogenesis. Interestingly, increased serum levels and genetic polymorphisms of various chemokines have been reported in athero-sclerosis and its clinical sequelae (Tables 3 and 4). This suggests that chemokines might not only play a prominent role in plaque initiation, progression and destabilization, but also in plaque rupture. Below, we will discuss the current insights on chemokine func-tion in atherothrombosis from an experimental as well as a clinical point of view and we shall review the relative contribution of each chemokine to atherothrombosis based on available experimental and clinical data.

MCP-1 / CCL2The CC chemokines are the largest family of chemokines, encompassing 28 different proteins. One of the CC chemokines, monocyte chemotactic protein-1 (MCP-1/CCL2), has been extensively studied with regard to atherosclerosis in animal models as well as in clinical studies. MCP-1 is expressed in macrophage-rich regions in human atheroscle-

Chemokines and Atherosclerotic Plaque Progression

31

rotic plaques and contributes to the recruitment of monocytes to the vessel wall23, 24.

Synonyms Receptor Source Actions

CCL2Small inducible cytokine A2, Monocyte Chemoattractant Protein-1 (MCP-1), SCYA2

CCR1 CCR2 MΦ/TL Mc, MΦ recruitment

CCL3 Small inducible cytokine A3, Macrophage Inflammatory Protein 1 alpha (MIP1α), SCYA3, G0S19-1

CCR1 CCR4 CCR5

MΦ/TL/NK/P

MΦ , Eo, N, BL recruitment

CCL4Small inducible cytokine A4, Macrophage Inflammatory Protein 1 beta (MIP1β), SCYA4

CCR1 CCR5 CCR8

TL/NK Mc, MΦ , Eo, TL recruitment

CCL5Small inducible cytokine A5, Regulated on Activation Normal T-cell Expressed and Secreted (RANTES), SCYA5

CCR1 CCR3 CCR4 CCR5

TL/P Mc, TL, Eo recruitment and adhesion

CCL11Small inducible cytokine A11, Eotaxin, Eosinophil chemotactic protein, SCYA11

CCR2 CCR3 CCR5

EC Mc, E, SMC recruitment, B activation

CCL13 Small inducible cytokine A13, Monocyte chemoattractant protein 4, SCYA4

CCR1 CCR2 CCR3 CCR5

EC

Eo, TL (Th2), Mc recruitment Eo, B activation

CCL17Small inducible cytokine A17, Thymus and activation-regulated chemokine (TARC), SCYA17

CCR4 CCR8

TL/EC/P/BL TL (Th2), Eo recruitment

CCL18

Small inducible cytokine A18, Dendritic cell chemokine 1, Macrophage inflammatory protein 4, Pulmonary and activation-regulated chemokine (PARC), SCYA18

CCR3 DC/Mc/MΦ/Eo,

TL, BL recruitmentMc/ MΦ activation

CCL19

Small inducible cytokine A19, Macrophage inflammatory protein 3 beta (MIP-3β), EBI1-ligand chemokine (ELC), SCYA19

CCR7 TL/SMC/ DC

TL(naïve, memory), BL, DC recruitment

CCL22

Small inducible cytokine A22, Macrophage-derived chemokine (MDC), Stimulated T cell chemotactic protein-1(STCP-1), SCYA22

CCR4TL/MΦ/EC/DC/BL

TL (Th2), DC, Eo, NK recruitment, Eo activation

Table 1 : CC Chemokines. BL = B Lymphocyte; B = Basophile; DC = Dendritic Cell; EC = Endothelial Cell; Eo = Eosinophile; MΦ = Macrphage; MC = Mast Cell; Mc = Monocyte; NK = Natural Killer Cell; N = Neutrophile; P = Platelet; SMC = Smooth Muscle Cell; TL = T Lymphocyte

As summarized in recent reviews22, 25, MCP-1 and its receptor CCR2 are most prominently involved in endothelial monocyte recruitment to sites of endothelial in-jury26. Also, increased levels of recombinant C-reactive protein (CRP), an in�lammatory marker shown to be up-regulated in atherosclerosis, was seen to promote monocyte in�lux by inducing CCR2 surface expression on circulating monocytes27. This �inding however should be addressed with care, while recently it has been suggested that this response is more likely due to proin�lammatory bacterial products which accompany re-combinant CRP28. Arterial MCP-1 expression levels were markedly elevated in response to hypercholesterolemia in primates and, in agreement, statin treatment directly inhib-ited MCP-1 mediated human monocyte recruitment in vitro23, 29. Several reports show an involvement of MCP-1 in vivo on the development of neo-intimal formation after vas-cular injury, neo-angiogenesis and also thrombosis. With regard to the latter, activated platelets were shown to augment monocyte recruitment by inducing MCP-1 synthesis

Chapter 2

32

by endothelial cells30. Moreover, MCP-1 and CCR2 de�icient mice as well as mice with a de�iciency in leukocyte CCR2 all showed decreased lesion formation but no differences in collagen content31-33, and blockage of MCP-1 activity by transfection of 7ND, an N- terminal deletion mutant of human MCP-1 gene, inhibited lesion initiation consider-ably34. Also, 7ND reduced monocyte in�iltration and caused an increase in plaque smooth cell and collagen content thereby promoting a stable plaque phenotype35. A similar effect was seen after administration of 11k2, a blocking antibody for MCP-1 and MCP-5, which reduced plaque area and concomitantly increased plaque collagen content36. Therefore, its relevance in plaque initiation seems undisputed, while its role in plaque progression and rupture is less clear. Viewed in that light it is very interesting to note that clini-cal studies could con�irm elevated MCP-1 serum levels in atherosclerotic complications such as acute myocardial infarction, unstable angina pectoris and ischemic stroke37, 38. Unfortunately, a causal in�luence of elevated MCP-1 serum levels in these conditions could not be shown. Also, a haplotype de�ined by the MCP1-2578G allele was associated with prevalent MI, providing genetic evidence on a role of MCP-1 in atherosclerosis39,

40. However, other studies did not detect any difference in MCP-1 serum levels between subjects with sub-clinical atherosclerosis and those without41,42. It is also shown that MCP-1 plays an important role in post infarction myocardial healing43. The increase in MCP-1 levels during myocardial infarction could therefore be more indicative of a MCP-1 driven myocardial healing response than a re�lection of plaque instability.

To conclude, MCP-1 is a very pleiotropic chemokine. While evidence for its role in plaque initiation seems compelling, its in�luence on plaque destabilisation is less evi-dent. With regard to the latter, a designated study on plaque stability on advanced le-sions would be desirable.

Platelet derived chemokines: RANTES and PF-4Not only do platelets play an essential role in hemostasis and thrombus formation, they also release various pro-in�lammatory substances upon activation, including chemo-kines. These chemokines are stored in α-granules and are mainly synthesized in the megakaryocytes but in part also endocytosed in situ by circulating platelets. The α-gran-ules contain an abundant range of chemokines which includes platelet factor 4 (PF-4), a member of the CXC chemokine family (CXCL4), and Regulated-on-activation,-normal-T-cell-expressed-andsecreted (RANTES, CCL5)44, 45. The latter, originally puri�ied as a product of activated T-cells and a ligand for CCR1 and CCR5, is a chemoattractant of T-lymphocytes, eosinophils and monocytes with potent endothelial arrest properties46-

48. PF-4 acts chemotactic for circulating monocytes and Th lymfocytes via its receptor CXCR349. It mediates E-selectin expression by endothelial cells, a process critically de-pendent on NF-κB activation50, and was seen to enhance the binding of oxidized low density lipoprotein (oxLDL) to endothelial cells and SMCs51, thereby participating in the onset of atheroma formation as well plaque progression. Upon activation, platelets se-crete RANTES and PF-4, triggering monocyte arrest after deposition of RANTES on the monocyte cell surface and on the activated endothelium52. Recently, Baltus et al. demon-strated that RANTES and PF-4 dose-dependently and synergistically induced endothe-lial monocyte arrest under conditions of �low which could be inhibited by Met-RANTES, a CCR1 and CCR5 antagonist, but also by a PF-4 blocking antibody48. The recent obser-vation that injection of ApoE-/- mice with activated platelets induced monocyte arrest and aggravated atherosclerotic lesion size may thus be partially attributable to platelet CCR5 and PF-413, 53. This was further substantiated by Veillard et al. showing that treat-ment of hyperlipidemic mice with Met-RANTES considerably inhibited atherogenesis resulting in reduced monocyte in�iltration and an increase in plaque collagen content, conferring a more stable plaque phenotype54. Interestingly however, mice de�icient in CCR5 were not protected against early plaque formation55. Recently, our lab showed that TAK-779, a HIV entry inhibitor which blocks CCR5 and CXCR3, impaired athero-genesis by blocking the recruitment of T cells (CD3+) to the plaque56, illustrative of the potential of chemokine receptor antagonists in plaque stabilizing strategies. In keeping

Chemokines and Atherosclerotic Plaque Progression

33

with the pro-atherothrombotic activity in mice models, PF-4 serum levels are elevated in peripheral vascular disease and CAD, possibly re�lecting enhanced platelet activity57. Pitsilos et al. observed a correlation between PF-4 expression and the lesion progres-sion stage from carotid artery plaques, linking chronic platelet activation and lesion development. They could detect PF-4 deposits on endothelium, macrophages and neo-vessels of human carotid arteries, even in early lesions, suggesting that PF-4 may play a major role throughout plaque development58. Studies in PF-4 knock-out models are eagerly awaited to verify this hypothesis. RANTES was not only shown to be expressed in human atherosclerotic plaques59, preliminary data show increased serum levels of RANTES in AMI and ACS patient groups as well, independent of other risk factors and in�lammatory markers60. However, serum levels were decreased in patients with stable CAD compared to controls41. Although the outcome of genetic association studies should be interpreted with care61, the RANTES G-403A polymorphism was independently as-sociated with CAD and this tendency was even more pronounced for subgroups of ACS and smokers, populations with increased platelet activation and in�lammation62.Al-though RANTES, and to a lesser extent PF-4, have a share in the onset of atherogenesis, their contribution to activity in plaque destabilization is less clear. The elevated serum levels of both chemokines in cardiovascular diseases may be due to platelet activation, yet their effect on plaque stability still has to be determined.

Eotaxin / CCL11Eotaxin (CCL11) was also shown to be present in human atherosclerotic plaques and its expression localized on SMCs22, 63. Originally described as a potent chemotractant and activator of eosinophils, a cell type which is only rarely observed in plaques, it acts by activating the CCR3 receptor on Th2 lymphocytes, dendritic cells, mast cells and other haematopoietic subsets. Moreover, CCR3 is expressed in macrophage-rich regions of atherosclerotic plaques63. Another interesting feature is the reported association of in-creased in�lammation, microvascular density and blood clotting in eotaxin-secreting tumors64. Recently, Emanuele et al. observed that increased plasma eotaxin levels cor-related with the severity of CAD, which contrasts however with studies by Mosedale et al. and Rothenbacher et al., who did not �ind any difference in serum eotaxin levels in CAD versus controls41, 42, 65. An A23T substitution in the CCL11 gene imparted an in-creased risk of myocardial infarction in a large prospective cohort of healthy American men66. Conversely, the –1328A/A promoter variant of the eotaxin gene appeared to con-fer protection against restenosis67. Given these con�licting data, the lack of solid data on eotaxin serum levels during acute ischemic events, and the lack of experimental data with genetically modi�ied mice, a potential in�luence of eotaxin on plaque destabiliza-tion remains to be proven.

MIP-1α / CCL3 AND MIP-1β / CCL4Macrophage in�lammatory protein-1α (MIP-1α/CCL3) and MIP-1β (CCL4) expression have been observed in human plaques, especially in the shoulder zone59, 68. They are secreted by a variety of cells. Amongst others, MIP-1α is released by activated platelets on the endothelium14. Whether or not MIP-1α levels are changed in atherothrombosis is unknown to date. Elevated levels of platelet derived IL-7 in stable and unstable an-gina pectoris were seen to be mediated by MIP-1α in a dose dependent manner and preliminary data from our lab indicate that MIP-1α levels are increased during acute myocardial infarction compared to a healthy control group (de Jager et al.; personal communication)69. These data show that MIP1α could play a role in plaque destabi-lization, a hypothesis further underscored by the fact that two MIP-1α receptors, i.e. CCR1 and 5, are both expressed on monocytes and endothelial cells, and the presence of the latter was observed in human vascular smooth muscle cells as well70. However, it is still unclear if elevated levels of MIP-1α are indicative of a causal relationship with myocardial infarction. For MIP-1β, aortic expression increased with lesion progression in mice36. Although no data on MIP-1β serum levels in human CVD are available, MIP-1β expression in peripheral mononuclear cells was increased in CAD patients compared to

Chapter 2

34

controls and atorvastatin could reduce its expression71. Even though data on the in�lu-ence of MIP-1α and MIP-1β in atherosclerosis are sparse, a role in plaque destabiliza-tion seems likely. Further experimental data are needed to verify this hypothesis.

IL-8 / CXCL8 AND GRO-α / CXCL1: CXC ChemokinesInterleukin 8 (IL-8, CXCL8) and growth related oncogene alpha (Gro-α, CXCL1, KC in mice) are related chemokines which both activate chemokine receptors CXCR1 and CXCR2 and contribute considerably to monocyte recruitment. Although IL-8 has ini-tially been linked to neutrophil activation and migration, it also appeared to be a potent mediator of �irm adhesion of monocytes to the vascular endothelium under �low condi-tions and thus is deemed to be a key determinant in the initiation of atherogenesis72. Likewise, Gro-α plays a signi�icant role in the arrest of monocytes to the endothelium through the activation of the β-2 integrin VLA-473. Circulating human monocytes ex-press CXCR1 and CXCR2 while IL-8 was abundantly present in macrophage-rich human plaques72. Leukocyte de�iciency of mIL8RH, the murine homologue of CXCR2, attenuated atherosclerosis in hyperlipidemic mice74. Another interesting feature of IL-8 is its ability to induce migration and proliferation of endothelial cells and vSMCs in vitro, pointing to a potential role in neovascularization75, 76. An elegant study of Simonini et al. con�irmed that IL-8 is angiogenic, via analysis of human coronary atherectomy tissue and the sub-sequent application of tissue derived IL-8 in a corneal sprouting assay, which may in the context of the plaque translate in expansive plaque growth and even plaque vulnerabil-ity75. In agreement, several lines of evidence further support an involvement of IL-8 in atherothrombosis. Not only were serum IL-8 levels recently found to be raised in stable coronary heart disease41, elevated levels were also noted in patients with acute myocar-dial infarction versus controls77, 78. A causal relationship of IL-8 and myocardial infarc-tion seems unlikely, while IL-8 levels could be enhanced upon activation by coagulation factor Xa in these patients77. Holm et al. provided mechanistic evidence showing that platelet-rich plasma, stimulated with oxLDL, enhanced IL-8 release by mononuclear cells in particular in CAD patients79. Also, peripheral blood mononuclear cells (PBMCs) derived from these patients and stimulated with oxLDL were seen to release more IL-8 and Gro-α than PBMCs from control patients. However, increased IL-8 levels could also be secondary to increased CRP levels, which is believed to cause a downstream upregu-lation of IL-880, but as mentioned before, this effect could be biased by bacterial side products of recombinant CRP28. These data are suggestive of a role of IL-8 in plaque de-stabilization but additional clinical trials as well as experimental research will have to shed further light on this matter. Conversely, no clinical data exist on Gro-α involvement during episodes of (acute) cardiovascular syndromes. Experimental data in ApoE*3 Leiden mice show that KC serum levels are raised on a high cholesterol diet compared to nontransgenic littermates, however no KC expression was seen in aortic lesions81. Therefore, Gro-α only seems to play a role in early lesion formation in response to el-evated plasma cholesterol levels, but a role in plaque destabilization seems less likely.

IP-10 / CXCL10IFNγ-inducible protein 10kDa (IP-10/CXCL10) expression has been detected in all three major atheroma-associated cell types in the plaque82. Although IP-10 has not yet been correlated with monocyte recruitment, it has been shown to participate in T-cell arrest via its receptor CXCR3 on activated endothelium. As already referred to by its name, IP-10 augments IFN-γ production by Th1 cells and may thus foster the in�lammatory response within the atherosclerotic vessel wall. Interestingly, Veillard et al. reported that initial lesion formation was inhibited in CCR2 ApoE double knockout mice83. How-ever, IP-10 is a potent inhibitor of tumour neo-angiogenesis as well and in agreement, neo-angiogenesis after myocardial infarction was found to be impaired after IP-10 ad-ministration84, 85. Therefore, IP- 10 in atherosclerotic plaques could act via attenuated angiogenesis, thereby hampering plaque progression and favouring plaque stability. Additional research has to be performed to exclude that IP-10 directly inhibits or pro-motes plaque progression in vivo, although recently, Cheng et al. showed that the de-

Chemokines and Atherosclerotic Plaque Progression

35

velopment of unstable lesions was characterized by upregulation of IP-10 during lesion initiation. While IP-10 serum levels were seen to be signi�icantly increased in a patient group with stable coronary heart disease (CHD) after adjustment for established risk factors41, a smaller study reported increased IP-10 expression in circulating T-cells of patients with stable versus unstable angina pectoris86. Although no conclusive data on IP-10 levels in acute ischemic syndromes are available, and as IP-10 mainly mediates T-cell function, a prominent role for IP-10 in plaque destabilization seems not unlikely. Currently, studies are underway to investigate this matter in patients with AMI.

Synonyms Receptor Source Action

CXCL1Small inducible cytokine B1, Growth regulated protein alpha, GRO-α, SCYB1

CXCR2 MΦ/P N recruitment and activation

CXCL4 Small inducible cytokine B4, Platelet factor 4, Oncostatin A, SCYB4 CXCR3 P L recruitment, EC, SMC

activation

CXCL8Small inducible cytokine B8, Interleu-kin 8, Monocyte-derived neutro-phil chemotactic factor, SCYB8

CXCR1CXCR2

EC/Eo/MΦ/SMC

N, Eo, TL , BL, B activation

CXCL10Small inducible cytokine B10, 10 kDA interferon-gamma inducible protein (IP-10), SCYB10

CXCR3 TL/SMC

Mc, TL, SMC recruitment and activation Angiostatic factor

CXCL11

Small inducible cytokine B11, Inter-feron-inducible T-cell alpha chemoat-tractant (ITAC), Interferon-gamma-in-ducible protein-9 (IP-9), SCYB11

CXCR3 EC/Mc/N TL (Th1), NK recruitment

CXCL12 Small inducible cytokine B12, Stromal cell-derived factor 1 (SDF-1), SCYB12 CXCR4 B/SMC

ECCD34+ Cell, Mc, TL, BL, MC recruitment

CXCL16

Small inducible cytokine B16, Scaven-ger receptor for phosphatidylserine and oxidized low density lipoprotein, Bonzo ligand, SCYB16

CXCR6MΦ/TL/SMC/DC

TL recruitmentfoam cell formation

CX3CL1Small inducible cytokine D1 , Fractalkine (FKN), neurotactin, SCYD1

CX3CR1 EC/N

Mc, SMC, N, MC recruit-ment, NK, L, P activation and adhesion, SMC prolife-ration

MIF Macrophage inhibitory factor CD74 L/EC MΦ, SMC activation

Table 2 : CXC, CX3C chemokines and other factors. BL = B Lymphocyte; B = Basophile; DC = Dendritic Cell; EC = Endothelial Cell; Eo = Eosinophile; MΦ= Macrophage; MC = Mast Cell; Mc = monocyte; NK = Natural Killer cell; N = Neutrophile; P = Platelet; SMC = Smooth Muscle Cell; TL = T Lymphocyte

SDF-1α / CXCL12Initially regarded as prime factor in the recruitment of progenitor cells from stroma, stromal cell-derived factor-1α (SDF-1α, CXCL12) appeared to be a rather pleiotropic chemokine, expressed by vSMCs, endothelial cells and macrophages of human athero-ma but not in the normal vessel wall52. Moreover, it is one of the few chemokines able to activate platelets, by interacting with CXCR4. SDF-1α is expressed constitutively and is a highly ef�icacious and potent mononuclear cell attractant in vivo87. Interestingly, SDF-1α was shown to display anti-in�lammatory activity by inhibiting leukocyte entry into in�lamed tissue. Furthermore, high concentrations of SDF-1α could have a potentially plaque stabilizing effect by in vitro inhibition of matrix metalloproteinase-9 (MMP-9) expression and enhancement of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) activity in peripheral blood mononuclear cells derived from patients with unstable angina pectoris (UAP)88. This was further underscored by the observation that SDF-1α levels were lower in patients with UAP vs. controls or stable angina pectoris patients88,

89. Recent publications mainly by Weber and co-workers focussed on the stimulatory

Chapter 2

36

role of SDF-1α in neointima formation after injury, regulating neointimal vSMC in�lux by recruitment of circulating progenitor cells90, which could be inhibited in vivo with an anti-SDF-1α antibody91. While progress on SDF-1α is limited by lethality of CXCR4 de- �iciency, other methods of gene manipulation are currently applied to unravel the pre-cise role of SDF-1α in atherothrombosis. For now, it seems that SDF-1α has a bene�icial effect on plaque stability, next to its more established stimulatory effect on mediating wound healing.

Chemokine Protein/RNA Expression

CCL2 Macrophage rich regions atherosclerotic plaquesElevated serum levels in AMI, UAP and ischemic stroke

CCL5 Increased serum levels in AMI and ACSDecreased serum levels in stable CAD

CCL11 Expression in atheromaControversy on enhanced serum levels

CXCL4 Increased serum levels peripheral vascular disease and CAD

CXCL8 Expressed in macrophage rich plaquesEnhanced serum levels in stable CAD and AMI

CXCL10 Enhanced serum levels in stable CAD

CXCL12 Decreased serum levels in UAP

CXCL16 Expression in atheroma

CX3CL1 Time dependent expression in plaquesEnhanced serum levels in ACS

MIF Expressed and active in advanced lesions

Table 3 : Expression of chemokines in human plaques and serum levels in CVD.

CXCL16CXCL16, also known as SR-PSOX, is an atypical chemokine. It is expressed in human atheroma, where it is localized on macrophages, endothelial cells and SMCs, but not in the unaffected vessel wall92-94. It exists as a membrane bound chemokine, at which the chemokine domain is attached to a transmembrane mucin stalk and in a soluble form after cleavage by ADAM-10 (a disintegrin and metalloprotease), its counter receptor is CXCR695. Soluble CXCL16 was reported to mediate migration of activated Tcells towards the intima as well as SMC proliferation. The transmembrane form serves as a scavenger receptor for oxLDL on macrophages and its expression is upregulated by IFN-γ, mediat-ing foam cell formation96, 97. Recently, CXCL16 was shown to affect aortic SMC function in an IL-18 and IFN-γ dependent manner, providing additional mechanistic support for the role of IL-18 in plaque destabilisation98, 99. Moreover, Kita and co-workers have de-monstrated that CXCL16 is a potent angiogenic factor100, fuelling the notion that CXCL16 could be involved in plaque neovascularisation and destabilization, although conclusive histopathological and in vivo data are still lacking. While evidence is accumulating that CXCL16 mediates T-cell in�lux, a role in monocyte arrest and in�lux remains to be estab-lished, though macrophages express CXCL16. Nevertheless, CXCL16 may reduce plaque stability by promoting foam-cell formation and subsequent release of proteases, as well as by mediating T-lymphocyte in�lux and to a lesser extent engage in expansive plaque growth. Lundberg et al. recently showed that the A181V polymorphism of CXCL16 gene enhanced coronary artery stenosis96. This observation, although tempting, is not direct-ly indicative of a role of CXCL16 in plaque vulnerability, since myocardial infarction is related to plaque instability rather than progressive stenosis. The fact that Sheikine et al. recently stated that CXCL16 might have an atheroprotective effect based on reduced plasma levels in UAP patients compared to controls, clari�ies that more experiments on CXCL16 on atherogenesis, plaque stability and scavenging capacities are needed in

Chemokines and Atherosclerotic Plaque Progression

37

order to draw de�initive conclusions101.

Chemokine Polymorphism Risk

CCL2 MCP-1-2578G ↑ AMI

CCL5 RANTES-G-403A ↑ CAD

CCL11 CCL11-A23T ↑ AMI

CXCL16 CXCL16-A181V ↑ Coronary Artery Stenosis

CX3CL1 CX3CL1-V249ICX3CL1-T280M

↑ CAD↓ CVD

Table 4 : Genetic polymorphisms.

Fractalkine/CX3CL1The CX3C family only contains a single member, Fractalkine or CX3CL1. While only recently identi�ied102, a vast body of evidence is already available on its in�luence in atherothrombosis as recently reviewed103. Fractalkine binds to its unique seven trans-membrane domain G-coupled protein receptor CX3CR1104. Unlike other chemokines, but similar to CXCL16, it is expressed as a membrane-bound protein, consisting of a 76-amino acid chemokine domain linked to a mucin-like stalk. It is highly expressed on the in�lamed endothelium, where it can serve as an adhesion molecule mediating se-lectin- and integrin-independent arrest of CX3CR1 expressing cells, like T-cells, speci�ic monocytes subsets and NK-cells103. Furthermore, tumour necrosis factor alpha convert-ing enzyme (TACE or ADAM-17) activity will release soluble fractalkine, which in turn acts as a chemo-attractant of the aforementioned cell types105. Membrane bound CX3CL augments the chemotactic activity of other chemokines. Once migrated, CX3CR1+ cells, among others, secrete IFN-γ, which enhances endothelial fractalkine expression, there-by forming a paracrine ampli�ication loop103. Hence, both forms support crucial steps in atherogenesis, and it is not surprising that fractalkine deeply impacts the pathobiology of atherosclerosis. Indeed, CX3CR1-/- mice on a hyperlipidemic background developed less atherosclerosis at the aortic root and displayed a more stable plaque phenotype106,

107. Yet, fractalkine knock-out mice on an ApoE-/- as well as LDLr-/- background showed reduction in brachiocephalic artery plaque formation but not in that of the aortic root108. Greaves et al. showed that fractalkine is expressed in human macrophages of advanced atherosclerotic lesions, in particular at neovascularized sites109. Lucas et al. extended this observation by stating that fractalkine expression in the plaque colocalizes with CX3CR1+ cells, most notably vSMCs. This observation was con�irmed in vitro by CX3CR1-mediated vSMC chemotaxis, which pleads for a plaque stabilizing effect of fractalkine110. Genetic polymorphism screening of different cardiovascular disease (CVD) popula-tions identi�ied CX3CR1 haplotype polymorphisms V249I and T280M. Intriguingly, the T280M mutation, which translated in impaired fractalkine activity, was independently found to confer protection for CVD in the Offspring Cohort of the Framingham Heart Study111, whereas the V249I polymorphism associated with an increased risk of CAD112,

113. In agreement, Niessner et al. showed that patients with CAD carrying the V249I but not the T280M allele were at elevated risk for acute coronary syndromes (ACS)114. Dur-ing ACS, plasma fractalkine levels were higher in V249I carriers as well, an observation which was recently con�irmed by Damas et al. showing increased fractalkine plasma levels in unstable CAD patients115. These data strongly suggest that the CX3CL1/CX3CR1 axis has important implications for plaque development and destabilization. A further sign of multifaceted activity is the fact that fractalkine can induce platelet activation and adhesion. Given its upregulation in atherosclerosis, fractalkine likely plays a dual role in atherothrombosis and subsequent ischemic symptoms116.

Chapter 2

38

MIF Macrophage inhibitory factor (MIF) is a very pleiotropic in�lammatory cytokine with a wide expression pattern117. While it shows strong structural resemblance with che-mokines and also was found to display chemotactic activity this cytokine is catego-rized as a chemokine like factor118. Its presence and activity was shown in the onset of atherogenesis as well as in advanced lesions of hypercholesterolemic rabbits and humans119, 120. It is expressed abundantly on the endothelium, where it induces ICAM-1 expression and on SMCs and macrophages, where it is also up-regulated after stimula-tion with oxLDL121. Pan et al. showed that MIF de�iciency considerably reduced athero-genesis in LDLr-/- mice122. Although no data are available on MIF serum levels in CVD, Schmeisser recently showed that advanced plaques express MIF, colocalizing mainly in areas of enhanced instability, such as increased mononuclear cell in�iltrates and neoves-sels123, Kong et al. suggested that this might be due to MIF regulated MMP-1 expression by vSMCs124. Burger-Kentischer et al. observed a decrease in i.e. ICAM-1, MMP-2 and CD40L expression in mice after treatment with an anti-MIF monoclonal antibody as well as a reduced intimal macrophage content, which however was only accompanied by a small reduction in plaque volume125. In contrast, Schober et al. did not �ind any signi�icant differences in plaque area after MIF blockade in a restenosis model, however MIF inhibition rendered a more stable intimal phenotype with reduced foam cell and increased SMC content126. Although this result, obtained in a wire injury model, should be extrapolated with caution, a signi�icant in�luence of the chemokine-like factor MIF in plaque destabilization appears plausible.

Other ChemokinesWhile there are around 50 chemokines, only a few additional chemokines have been implicated in atherothrombosis, however their exact role in plaque rupture and its sequelae still has to be determined. A brief description of recent �indings is that MIP-3β (CCL19) and MCP4 (CCL13) were shown to be expressed in human plaques127, 128. Furthermore, pulmonary and activation regulated chemokine (PARC, CCL18) could be detected in human plaques, colocalizing with CD68+ macrophages127. The related thymus-and activation-regulated chemokine (TARC, CCL17) and macrophage derived chemokine (MDC, CCL22) are also associated with macrophages in advanced plaques, especially at sites with increased neovascularization suggesting that these chemo-kines could be involved in monocyte recruitment or macrophage activity as well109 . Also, TARC and MDC are able to induce platelet aggregation via their shared receptor CCR4, which could be inhibited by a monoclonal antibody against CCR414, 129. Howev-er in patient cohorts, correlative data of PARC, TARC or MDC serum levels or genetic polymorphisms in CVD are still lacking, which renders their local or systemic role in atherothrombosis and plaque rupture unknown. Finally, one of the more exotic ligands of CXCR3 seems to be involved in atherogenesis as increased IFN-inducible T cell alpha chemoattractant (ITAC,CXCL11) serum levels have been demonstrated to correlate with the development of transplant coronary artery disease130. Histological and expression studies on chemokines in atheroma are of great importance. Accordingly, the contribu-tion of a majority of functional chemokines in atherogenesis is still not fully understood, thereby warranting chemokine pro�iling studies with speci�ic patient groups as well as experimental research in representative mouse models for atherosclerosis or coronary ischemia after pharmacological or genetic intervention in chemokine function. These studies will reveal whether and to what extent the described chemokines are involved in atherogenesis or injury repair after myocardial infarction, whether they are associ-ated as a biomarker or as a speci�ic culprit for plaque rupture and, if so, whether these chemokines are instrumental in disease progression and constitute relevant therapeu-tic targets for intervention.

Therapeutic options for plaque stabilization; current advancesStudies in mice genetically de�icient for a single chemokine or receptor have not only

Chemokines and Atherosclerotic Plaque Progression

39

increased our understanding of their role in atherosclerosis, plaque stability and re-stenosis but also helped to identify new targets for future intervention. While some chemokines, such as MCP-1, Fractalkine and RANTES appear to aggravate the process, others are believed to have an atheroprotective effect. To complicate matters, the activ-ity of some chemokines was shown to be stage dependent. Given their dedicated role in in�lammation and high selectivity, chemokines are very interesting therapeutic targets for intervention in atherothrombosis, while their GPCRs are generally favourable drug targets. This notion prompted pharmaceutical companies to put considerable effort in the design of synthetic chemokine receptor antagonists for other in�lammatory diseas-es like multiple sclerosis, HIV infection and rheumatoid arthritis. Various compounds already have entered clinical phase II and in fact CCR1 and CCR5 receptor antagonists already advanced to phase III trials for HIV/AIDS and rheumatoid arthritis131. Despite the apparent promise, to our knowledge, trials to validate the ef�icacy of chemokine receptor antagonists in atherothrombosis or related disorders of various chemokine candidates have not been initiated to date.

Illustratively, injection of Met-RANTES, a combined CCR1 and CCR5 antago-nist, halted the progression of atherosclerosis in animal models54, while our group has recently demonstrated attenuated lesion formation in vivo after treatment with TAK-779, originally designed as an HIV entry inhibitor and a CXCR3 and CCR5 antagonist for Th1 cells56. However, a plaque stabilising effect of these compounds in already existent atherosclerosis remains to be determined. Another technical drawback is that chemo-kine antagonists quite often lack species cross-reactivity mandatory for extrapolation from an experimental to a clinical setting132. Apart from synthetic antagonists, which may lack receptor speci�icity or display an unfavourable activity pro�ile, another effec-tive approach involves the use of viral chemokine antagonists and chemokine mutants. Its ef�icacy was recently demonstrated by Bursill and co-workers showing dramatically reduced lesion area, macrophage content and cell proliferation in a diet induced athero-sclerosis model as well as in a vein graft atherosclerosis model after in vivo administra-tion of the broad spectrum CC chemokine binding protein 35K133, 134. On the other hand, no differences in smooth muscle cell or collagen content were seen. Pyo et al. showed a reduction in wire induced neo-intimal hyperplasia in mice expressing M3, a herpesvirus protein that binds and inhibits all 4 chemokine subgroups135. However, M3 was tested in a mouse model of restenosis for its effect on intimal hyperplasia, a process which, unlike atherosclerosis, is critically dependent on vSMC proliferation. Both 35K and M3 act on a broad range of chemokines, thereby precluding the opportunity of selective chemokine inhibition and enhancing the chance of adverse side effects in humans.

Recently, our lab was able to reduce atherogenesis and induce a more stable plaque phenotype using a novel vaccination strategy against IL-12136. Although no such attempts have been undertaken for the blockade of speci�ic chemokines, it may be very well suited for targeted inhibition of the chemokine system. In addition, focal delivery is very interesting, as chemokines are at the very heart of immuno-mobilisation and recruitment. Receptor antagonists are suited for site directed topical application at the aimed lesion site via for instance drug-eluting coronary stents, local viral transfer or gene silencing using siRNA137. Plausibly, systemic intervention in chemokine function, although desirable in the context of wide-spread atherosclerosis, could render consi-derable side effects. A second strategy to confer speci�icity is co-therapy: while most chemokines act in concert as heterodimers interacting with functionally coupled recep-tors, anti-dimerizing agents could yield another viable therapeutic possibility138.

Interestingly, apart from their expected therapeutic targets, current cardiovas-cular drugs appear to modulate chemokine activity as well. For instance, it has become increasingly clear that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase or statins exert antiin�lammatory properties, independent of their lipid lowering effect. They are shown to decrease chemokine secretion by inhibition of the

Chapter 2

40

geranyl geranylation of GTP-binding intracellular signalling proteins, including Rho, Ras and Rac139. By illustration, Pitavastatin (NK-104) was seen to suppress IL-8 production in human endothelial cells 80. Still, although statin use has proven to promote a stable plaque phenotype, both in animal models and humans, the contribution of chemokine modulation by statins to this effect remains to be established140. However, statins are very pleiotropic and various mechanisms may underlie their plaque stabilizing effects, obscuring to the contribution of statin mediated chemokine inhibition to plaque stabili-zation. Other chemokine modulating drugs include aspirin, which was demonstrated to inhibit TNF-α induced MCP-1 and IL-8 expression and protein levels in endothelial cell as well as monocyte adhesion and transmigration in vitro141. Finally, the angiotensin AT-1 receptor antagonist irbesartan, apart of its anti-hypertensive effects, reduced lesion progression in part by inhibiting MCP-1 and Gro-α expression142, fuelling the notion that angiotensin II is a mediator of vascular wall in�lammation143.

The main drawback for clinical trials on chemokine intervention in cardio-vascular disorders is the lack of a reasonable endpoint. As for now, advanced imaging techniques seem most eligible to evaluate the effect of chronic chemokine inhibition in plaque stability. For the evaluation of chemokine therapy in ACS, while it could serve to stabilize in�lammation, reduce platelet activity and induce plaque stability, a clear read-out in serum is favoured. Therefore, regardless of the potential of chemokine therapy, the search for a biomarker speci�ically correlating with lesion severity or plaque desta-bilization which could very well involve a certain chemokine, is ongoing and of utmost importance. Such a biomarker is applicable for the strati�ication of high risk patients as well as for the tailoring of athero-protective and regressive therapy.

Figure 1: Schematic overview of mechanisms in atherothrombosis in which chemokines are seen to participate. (s)FKN = (soluble) fractalkine

Conclusion and future directions/perspectivesOver the last years, our understanding of the role of various chemokines to the patho-

Chemokines and Atherosclerotic Plaque Progression

41

Rupture

ThrombusSDF1α

Platelet Activation RANTESPF4

Chemotaxis

Shoulder RegionNecrotic core

Circulating monocytes

Media

TIMP

TACE

sFKN Lumen

FKN

MMP-9

vSMC

Migration

IntimaIL-8FKN

Neovascularization

RANTES

PF-4

SDF1α

Fibrous cap

Necrotic core

Endothelium

genesis of atherosclerosis and plaque progression has increased tremendously. While the contribution of the whole chemokine system in immunology and atherosclerosis is very complex and overlapping, it is most likely that chemokines, either in concert of after heteromerization, may have their own distinctive role at speci�ic stages of dis-ease progression. This speci�icity therefore puts the chemokines and their receptors in the therapeutic spotlight. Experimental studies in animal models have already helped to identify a number of interesting targets for intervention in atherosclerotic plaque growth or thrombotic plaque rupture, and it is expected that this selection will rapidly expand in the near future. The fact that chemokine receptors are G-coupled protein receptors render them eligible for drug design, and indeed for many chemokine recep-tors potent and rather selective antagonists have been developed. Still, validation of their potential in anti-atherothrombotic therapy is hampered by the lack of a good sur-rogate marker for this disease and one of the reasons why short term clinical trials in humans currently remain cumbersome. Nevertheless, knockout models continue to be developed and dozens of chemokine receptor antagonists have already been tested in other immune disorders. In addition, taking the numerous ways of drug administration and delivery into account, science awaits an exciting increase in our understanding of this complex disease, enabling the development of speci�ic chemokine-targeted athero-regressive therapies besides current lipid lowering strategies.

AcknowledgementsA.O. Kraaijeveld was supported by the Molecular Cardiology Program of the Netherlands Heart Foundation (grant nr. M93.001). S.C.A. de Jager was supported by the Netherlands Heart Foundation (grant nr. 2003T201). E.A.L. Biessen and J.W. Jukema are Established Investigators of the Netherlands Heart Foundation (grants 2003T201 and 2001D032 respectively).

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Department of Cardiology and Einthoven Laboratory of Experimental Vascular Medicine, Leiden University Medical Center, the Netherlands. Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug research, Leiden University, the Netherlands. Department of Paediatric Immunology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, the Netherlands. School of Molecular & Biomedical Science, The University of Adelaide, Australia. Division of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden, the Netherlands. Div. of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden, the Netherlands.Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, the Netherlands. Department of Pathology, Academic University Hospital Maastricht, the Nether-lands

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